1. Field of the Invention
The present invention relates generally to the field of transformed cells. More particularly, it concerns Ras-transformed cells and provides compositions and methods for selectively inhibiting the growth of cells with ras mutations.
2. Description of the Related Art
In normal cells, Ras, a plasma membrane-associated 21 kDa guanine nucleotide-binding protein, is involved in important cellular events on the molecular level, particularly signal transduction events associated with both normal and oncogenic cell growth and differentiation (Barbacid, 1987). In normal cells, Ras is activated following the stimulation of fibroblasts by growth factors (Lu and Campisi, 1992) the stimulation of hematopoietic cells by the cytokines interleukin-3 and granulocyte-macrophage colony-stimulating factor (Satoh et al., 1991), and the stimulation of T lymphocytes by the cytokine, interleukin-2 and antibodies to the T cell antigen receptor and the alternative activation molecule, CD2 (Downward et al., 1990; Graves et al., 1992). The exact function of activated Ras in normal cells has not been defined. However, its importance has been inferred from studies demonstrating that depletion of normal levels of Ras or neutralization of Ras activity interferes with cell growth.
The Ras protein has also been associated with oncogenic cell growth. For example, about 20% of all human tumors have been found to have a mutation that activates a cellular ras proto-oncogene (Barbacid, 1987); these mutations are particularly prevalent in common cancers such as adenocarcinomas of the colon (Fearon et al., 1990) and pancreas (Almaguera et al., 1988), as well as in non-small cell lung carcinoma (Mitsudomi, et al., 1991). Oncogenic forms of Ras are constitutively active, generally as the result of a point mutation that leads to the accumulation of GTP-bound Ras (Grand et al., 1991).
Membrane association of Ras is important for in vitro transforming activity and is, in normal cells, dependent on post-translational modification of the carboxy-terminus by sequential farnesylation, proteolysis and carboxymethylation (Willumsen et al., 1984; Hancock et al., 1989 cell). In addition, either a polybasic domain or palmitoylation is necessary for localization to the plasma membrane (Hancock et al., 1990). Of this step-wise series of post-translational modifications, initial farnesylation appears to be a requisite event for both membrane association and in vitro transformation by oncogenic Ras (Jackson, et al., 1990; Hancock et al., 1990). Interference with post-translational farnesylation of Ras prevents subsequent membrane localization (Willumsen et al., 1984; Jackson et al., 1990; Karo et al., 1992).
Recent studies have elucidated the biochemical processes that convert cytosolic Ras into its membrane-associated form. In the initial farnesylation step, cytosolic farnesyl-protein transferase utilizes farnesyl pyrophosphate to modify cysteine-186 in the carboxy-terminal sequence of cytosolic Ras. Inhibition of the synthesis of farnesyl pyrophosphate, by blocking the formation of the precursor mevalonate with lovastatin, prevents post-translational processing of Ras and subsequent membrane localization. Theoretically, therefore, lovastatin may be able to inhibit proliferation of Ras-transformed cells by blocking farnesylation, if the cell growth is Ras-dependent. However, other cellular effects of the inhibition of mevalonate synthesis by lovastatin interfere with its usefulness in determining the role of farnesylation of Ras and also limit its potential ability to inhibit the growth of Ras-transformed cells selectively.
Lovastatin inhibits the proliferation of all cells by preventing the synthesis of at least two essential products of mevalonate metabolism (FIG. 1). One of these products has been clearly shown to be cholesterol. The other product has only been defined indirectly and has not been identified. Thus, when the synthesis of mevalonate is blocked with lovastatin or related specific inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cell proliferation is completely prevented. Neither trace quantities of mevalonate nor its end-product cholesterol is individually able to restore growth. However, when both are added together, proliferation is totally restored. These studies provide evidence that both cholesterol and non-sterol products derived from mevalonate are each required for normal cell growth. It should be noted that neither the number nor the identity of the non-sterol product(s) of mevalonate required for cellular proliferation has been defined. Furthermore, whether the role of the mevalonate product(s) in the proliferation of transformed cells is different than the role in non-transformed cells has not been clarified.
The most preferred forms of cancer therapy are designed to control the growth of malignant cells, while permitting proliferation and function of normal cells. However, many chemotherapeutic agents and cancer-directed pre- and post-surgery treatments destroy or otherwise severely compromise the non-cancerous population of cells in the patient. Although the rational behind such therapies is that any remaining non-oncogenic, healthy cells in the patient will repopulate and restore the patient to an improved immunological state, it is, of course, well-known that all current treatment methods for cancer are far from satisfactory and that improved therapeutic regimens are needed.